Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this ...disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.
van Agtmaal et al. demonstrate that excision of the expanded (CTG⋅CAG)n repeat from the DMPK gene in myotonic dystrophy patients can be reliably achieved by dual CRISPR/Cas9 cleavage at either side of the repeat and that this offers a strategy to adjust anomalous effects of expanded DMPK transcripts in myoblasts.
Abstract
Background
Myotonic dystrophy type 1 (DM1) is an incurable multisystem disease caused by a CTG-repeat expansion in the DM1 protein kinase (
DMPK
) gene. The OPTIMISTIC clinical trial ...demonstrated positive and heterogenous effects of cognitive behavioral therapy (CBT) on the capacity for activity and social participations in DM1 patients. Through a process of reverse engineering, this study aims to identify druggable molecular biomarkers associated with the clinical improvement in the OPTIMISTIC cohort.
Methods
Based on full blood samples collected during OPTIMISTIC, we performed paired mRNA sequencing for 27 patients before and after the CBT intervention. Linear mixed effect models were used to identify biomarkers associated with the disease-causing CTG expansion and the mean clinical improvement across all clinical outcome measures.
Results
We identified 608 genes for which their expression was significantly associated with the CTG-repeat expansion, as well as 1176 genes significantly associated with the average clinical response towards the intervention. Remarkably, all 97 genes associated with both returned to more normal levels in patients who benefited the most from CBT. This main finding has been replicated based on an external dataset of mRNA data of DM1 patients and controls, singling these genes out as candidate biomarkers for therapy response. Among these candidate genes were
DNAJB12
,
HDAC5
, and
TRIM8
, each belonging to a protein family that is being studied in the context of neurological disorders or muscular dystrophies. Across the different gene sets, gene pathway enrichment analysis revealed disease-relevant impaired signaling in, among others, insulin-, metabolism-, and immune-related pathways. Furthermore, evidence for shared dysregulations with another neuromuscular disease, Duchenne muscular dystrophy, was found, suggesting a partial overlap in blood-based gene dysregulation.
Conclusions
DM1-relevant disease signatures can be identified on a molecular level in peripheral blood, opening new avenues for drug discovery and therapy efficacy assessments.
Disease burden of myotonic dystrophy type 1 Landfeldt, Erik; Nikolenko, Nikoletta; Jimenez-Moreno, Cecilia ...
Journal of neurology,
04/2019, Letnik:
266, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Objective
The objective of this cross-sectional, observational study was to investigate the disease burden of myotonic dystrophy type 1 (DM1), a disabling muscle disorder.
Methods
Adults with DM1 ...were recruited as part of the PhenoDM1 study from Newcastle University (Newcastle upon Tyne, UK). Disease burden data were recorded through the Individualized Neuromuscular Quality of Life (INQoL) questionnaire. Results were examined by sex and clinical variables e.g. the six-minute walk test (6MWT), the Mini Mental State Examination, and estimated progenitor and modal allele CTG repeat length.
Results
Our sample consisted of 60 patients with DM1 (mean age: 45 years; 45% female). Muscle weakness and fatigue constituted the two most common disease manifestations, reported by 93% and 90% of patients, respectively, followed by muscle locking (73%). Most patients (> 55%) reported feeling anxious/worried, depressed, frustrated, and/or having low confidence/self-esteem, 23% and 33% indicated substantial impairment of daily and leisure activities, respectively, and 47% did not work as a consequence of the disease. Estimated progenitor CTG length corrected by age correlated surprisingly well with INQoL scores. Differences by sex were generally minor.
Conclusion
We show that DM1 is associated with a substantial disease burden resulting in impairment across many different domains of patients’ lives, emphasizing the need for a holistic approach to medical management. Our results also show that the INQoL records relevant information about patients with DM1, but that further investigation of the psychometric properties of the scale is needed for meaningful interpretation of instrument scores.
Genetically unstable expanded CAG·CTG trinucleotide repeats are causal in a number of human disorders, including Huntington disease and myotonic dystrophy type 1. It is still widely assumed that DNA ...polymerase slippage during replication plays an important role in the accumulation of expansions. Nevertheless, somatic mosaicism correlates poorly with the proliferative capacity of the tissue and rates of cell turnover, suggesting that expansions can occur in the absence of replication. We monitored CAG·CTG repeat instability in transgenic mouse cells arrested by chemical or genetic manipulation of the cell cycle and generated unequivocal evidence for the continuous accumulation of repeat expansions in non-dividing cells. Importantly, the rates of expansion in non-dividing cells were at least as high as those of proliferating cells. These data are consistent with a major role for cell division-independent expansion in generating somatic mosaicism in vivo. Although expansions can accrue in non-dividing cells, we also show that cell cycle arrest is not sufficient to drive instability, implicating other factors as the key regulators of tissue-specific instability. Our data reveal that de novo expansion events are not limited to S-phase and further support a cell division-independent mutational pathway.
Objective
The objective of this longitudinal, observational study was to investigate change over time in ability to perform activities of daily living in myotonic dystrophy type 1 (DM1).
Methods
...Adults with genetically confirmed DM1 were recruited as part of the PhenoDM1 study in the UK. Data on activities of daily living were recorded through the DM1-Activ
C
at baseline and a follow-up visit after 12 (± 3) months. A subset of patients had advanced genetic testing to determine the size of the progenitor allele.
Results
Our sample comprised 150 patients with DM1 (mean age: 45 years; 52% female). Mean follow-up was 383 days. Mean DM1-Activ
C
total score at baseline was 71.24 (95% confidence interval 67.77–74.71) and at the follow-up visit 69.04 (65.54–72.54). Approximately 43% of patients had a lower score at the follow-up visit (indicating a decreased ability to perform activities of daily living), 24% a higher score (indicating an increased ability), and 33% the same score at baseline and follow-up. The mean annual change in the DM1-Activ
C
total score, estimated at − 2.06 (− 3.54 to − 0.59), was significantly related to patients’ baseline score, but not sex, disease duration, timed test results, or cytosine-thymine-guanine repeat length.
Conclusions
Change over time in ability to perform activities of daily living as recorded through the DM1-Activ
C
varies substantially between patients with DM1. Our data contribute to the understanding of the natural evolution of the disease, and should be helpful to inform the design of future trials based on the DM1-Activ
C
.
Mutation at the human minisatellites MS32, MS205 and MS31A has been investigated by characterizing mutant alleles in pedigrees and in the case of MS32 by direct analysis of mutant molecules in single ...sperm. Most mutations at all three loci are polar, involving the preferential gain of a few repeat units at one end of the tandem repeat array. Incoming repeats can be derived from the same allele or the homologous chromosome, through they are frequently rearranged during mutation. Lack of exchange of flanking markers suggests the involvement of complex conversion-like events in the generation of mutant alleles. At MS32, high frequency mutation processes in sperm appear to be largely germline specific and to occur at a constant rate irrespective of allele size. Together with mutational polarity, this implies that germline instability is controlled by elements outside the tandem repeat array.
Objectives
The objective of this cross‐sectional, observational study was to investigate performance of activities of daily living in patients with myotonic dystrophy type 1 (DM1).
Materials and ...Methods
Adults with genetically confirmed DM1 were recruited from Newcastle University (Newcastle upon Tyne, UK) and University College London Hospitals NHS Foundation Trust (London, UK). Data on activities of daily living were recorded through the DM1‐ActivC (scale scores range between 0 and 100, where a higher/lower score indicates a higher/lower ability).
Results
Our sample comprised 192 patients with DM1 (mean age: 46 years; 51% female). Patients reported most difficulties with running, carrying and putting down heavy objects, and standing on one leg, and least difficulties with eating soup, washing upper body, and taking a shower. Irrespective of the disease duration (mean: 20 years), most patients were able to perform basic and instrumental activities of daily living (eg personal hygiene and grooming, showering, eating, cleaning and shopping), with the exception of functional mobility/transfer tasks (eg walking uphill and running). The mean DM1‐ActivC total score was estimated at 71 (95% CI: 68‐74). Estimated progenitor cytosine‐thymine‐guanine repeat length and age explained 27% of the variance in DM1‐ActivC total scores (P < .001).
Conclusions
We show that DM1 impairs performance of activities of daily living, in particular those requiring a high degree of muscle strength, stability and coordination. Yet, across the evolution of the disease, the majority of patients will still be able to independently perform most basic and instrumental activities of daily living.
An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including ...Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with a phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels ofinstability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis-acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats.
In order to characterize the dynamics of CTG repeat instability in somatic and germline tissue from myotonic dystrophy (DM) males we have used small pool polymerase chain reaction (PCR) in a detailed ...quantitative analysis of repeat length variation. We demonstrate that the heterogeneous smear of CTG repeats observed in DM patients using standard analyses is comprised of multiple unresolved bands that may be dissected into discrete length alleles derived from single cells using single molecule PCR techniques. Analysis of somatic tissues demonstrates a bias toward increasing allele length and a lower boundary below which variant alleles are rare, consistent with a highly directional expansion pathway in the soma. Two sperm samples show extensive variation and a size increase bias, concordant with the phenomenon of anticipation. In addition, sperm analysis shows that large contractions, including reversions into the normal size range, are restricted to the germline. Detailed analysis of intergenerational 'reductions' paternally transmitted to two offspring suggests that some apparent reductions may be artifacts of somatic expansion in the parent. Our data indicate that in addition to germline variation, substantial somatic expansion can also contribute to the intergenerational differences usually observed in DM.
Myotonic dystrophy type 1 (DM1) is one of a growing number of inherited human diseases whose molecular basis has been implicated as the expansion of a trinucleotide DNA repeat. Expanded ...disease-associated alleles of >50 CTG repeats are unstable in both the germline and soma. Expansion of the unstable alleles over time and variation of the level of mutation between the somatic tissues of an individual are thought to account at least partially for the tissue specificity and progressive nature of the symptoms. We previously generated a number of transgenic mouse lines containing a large expanded CTG repeat tract that replicated a number of the features of unstable DNA in humans, including frequent sex-specific changes in allele length during intergenerational transmission. Small length change mutations were apparent in the somatic tissues of young mice in all of the lines generated, but the gross instability observed in human DM1 patients was not replicated. We now show that in one of the lines, Dmt -D, spectacular, expansion-biased, tissue-specific instability is observed in older mice. The highest levels of instability were detected in kidney with gains of >500 repeats, representing a tripling of allele length, in some cells. Mosaicism accumulated in an age-dependent manner, but the tissue specificity did not obviously correlate with cell turnover. Such gross somatic mosaicism was not observed in three other lines examined, further emphasizing a role for flanking DNA in modulating repeat stability.
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